Cell culture of hepatitis A virus

ABSTRACT

Hepatitis A virus is attenuated in virulence by in vitro passage in susceptible cell cultures, without prior passage in a subhuman primate. This process results in a live, attenuated hepatitis A virus vaccine suitable for human disease prevention.

This application is Continuation of application Ser. No. 08/016,486,filed Feb. 10, 1993, now U.S. Pat. No. 5,514,376; which is acontinuation of application Ser. No. 07/712,365, filed Jun. 10, 1991,now abandoned; which is a Continuation of application Ser. No.07/126,458, filed Nov. 27, 1987, now abandoned; which is aContinuation-In-Part of application Ser. No. 06/609,677, filed May 14,1984, now U.S. Pat. No. 5,021,348; which is a Continuation ofapplication Ser. No. 06/437,095 filed Oct. 27, 1982, now abandoned;which is a Continuation-In-Part of application Ser. No. 06/071,648,filed Sep. 4, 1979, now abandoned.

BACKGROUND OF THE INVENTION

The in vitro cultivation of hepatitis A virus (HAV) eluded all attemptsfor several decades. The modern era of advances in understanding HAV,which led eventually to in vitro cultivation of the virus, was initiatedby the successful transmission of HAV to marmosets Deinhardt et al., J.Exp. Med. 125, 673 (1967), Mascoli et al., Proc. Soc. Exp. Biol. Med.142, 276 (1973) and Provost et al., Proc. Soc. Exp. Biol. Med. 142,1257, (1973)!, by the characterization of HAV from marmoset serum andliver Provost et al., Proc. Soc. Exp. Biol. Med. 148, 532 (1975)!, andby the detection of HAV in the stools of infected humans by immuneelectron microscopy (IEM), Feinstone et al., Science 182, 1026 (1973)!.

Progress in cultivating and characterizing the CR 326 strain of HAV inmarmosets led to its definition as a picornavirus Provost et al., Proc.Soc. Exp. Biol. Med. 148, 532 (1975)!, to the development of serologicassays for HAV antigen and antibody Provost et al., Proc. Soc. Exp.Biol. Med. 148, 962 (1975) and Miller et al., Proc. Soc. Exp. Biol. Med.149, 254 (1975), and to the development of potent preparations of HAVfrom infected liver of marmosets Provost et al., Proc. Soc. Exp. Biol.Med. 148, 532 (1975), and Provost et al., Proc. Soc. Exp. Biol. Med.155, 283 (1977)!. Such preparations contained about 10⁹ fifty percentmarmoset infectious doses per gram of liver tissue. These findingsprovided the background for the successful propagation of HAV in cellculture first reported by Provost et al., Proc. Soc. Exp. Biol. Med.160, 213 (1979).

A disadvantage of the foregoing methods is that they require the use ofprimates that are not only expensive but in short supply and difficultto obtain. An in vitro cell culture system which did not require viruspassage in vivo in a subhuman primate would be a significant advance inthis art.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide, by in vitro cellculture of hepatitis A virus, an attenuated hepatitis A virus suitablefor use as a vaccine. Another object is to provide a method forpropagation of hepatitis A virus which does not require virus passage invivo in a subhuman primate. A further object is to provide a method forthe preparation of hepatitis A antigen for use in vaccines and as adiagnostic antigen. Still another object is to provide an in vitromethod for attenuating the virulence of hepatitis A antigen. These andother objects of the present invention will be apparent from thefollowing description.

SUMMARY OF THE INVENTION

Live attenuated HAV modified by passage in susceptible cell cultures,without prior passage in vivo in a subhuman primate, so as to replicatein humans and induce an immune response to protect against typicalhepatitis A infection without itself causing any significant symptoms ofhepatitis A disease.

DETAILED DESCRIPTION

The present invention relates to the in vitro cell culture propagationof hepatitis A virus and, more particularly, to in vitro cell culturepropagation of hepatitis A virus wherein the inoculum is a humanclinical specimen containing the virus.

The present invention provides a method for propagating hepatitis Avirus wherein passage of the virus in a susceptible non-human primateprior to in vitro cell culture is not required.

According to the present invention a clinical specimen containinghepatitis A virus, e.g. stool extract, saliva, urine, or blood, is usedas inoculum to infect a susceptible in vitro cell culture. The cellculture may be formed of primary, or low passage, or continuouslycultivated, or transformed cells derived from kidney or liver of humanor non-human primate origin or diploid fibroblast cells derived fromhuman or non-human primate lung tissue. Specific examples of suitablecell cultures are low passage cell cultures derived from fetal (FRhK6)or newborn kidney cells from rhesus monkey, cynomolgus monkey, orcercopithecus monkey.

The inoculated cell culture is incubated at a temperature in the rangeof from about 28° to about 39° C., preferably at from about 32° to about35° C. for a period of time until positive results are obtained for thepresence of hepatitis A antigen; time required may vary from 3-200 days,generally from about 25 to about 100 days. The incubation is carried outin the presence of a nutrient medium which maintains viability of thecells at temperatures permitting propagation of the virus in the cellculture. The nutrient medium may be, for example, Eagle's MinimumEssential Medium (EMEM), Williams Medium E, Medium 199, Dulbecco'sModified Eagle's Medium, RPMI Media or Basal Medium Eagle with fromabout 0.5 to about 10% fetal calf serum. The cultures are subsequentlyharvested and serial passages of the viral agent are carried out.

The virus may be additionally propagated by serial passages in diploidfibroblast cells derived from human or non-human primate lung tissue.Propagation is effected through at least about 2 serial passages in thediploid fibroblast cells such as, for example, WI-38 or MRC5 cell lines.Preferably propagation is effected through from about 5 to about 100passages, and most preferably from about 5 to about 40 passages, in thediploid fibroblast cells. Most preferably, prior to passage in thediploid fibroblast cells the virus is propagated in a cell-culturederived from primary or low passage, continuously cultivated ortransformed cells derived from kidney or liver of human or non-humanprimates. Propagation is effected through from about 2 to about 50passages in the kidney or liver of human or non-human primates.

This invention allows disease diagnosis by direct virus isolation.Further, it allows the in vitro cultivation of the virus in adequatequantity and with appropriate properties for the preparation of live,attenuated or killed, inactivated human vaccines or for use as adiagnostic antigen. A live, attenuated vaccine virus is a virus that isable to replicate in a host animal or a human being without producingdisease symptoms while maintaining the capacity to induce an immuneresponse and thus to provide protection against hepatitis A disease. Akilled, inactivated vaccine virus is a virus that is unable to replicatein cell culture, a host animal or a human being but which, uponinjection in sufficient mass, is capable of inducing an immune responseand thus protecting against hepatitis A disease.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

A. A 20 percent extract of human stool in PBS was prepared from acutelyill human hepatitis A patients and clarified by centrifugation. It gavean immune adherence hemagglutination titer of 1:16 for hepatitis Aantigen. It was further diluted 1:5 in cell culture medium containing0.5% fetal calf serum and filtered through a 0.45μMillipore filter. Oneml of this material was inoculated per 25 cm² flask of fetal rhesuskidney (FRhK6) cells. The inoculum was left in place for 4 hours andthen removed. Five ml of EMEM containing 0.5% fetal calf serum plusneomycin and glutamine were added per flask and the medium removed andrenewed at 5-7 day intervals with incubation at 32°-35° C. Coverslipswere removed periodically and examined by the direct immunofluorescencetechnique (Provost et al., P.S.E.B.M., 160, 213-221, 1979) for HAV. Suchexamination was essentially negative until day 33 post inoculation, whenthe first clear positive results were obtained. The cultures weresubsequently harvested at day 37 and the cells disrupted byfreeze-thawing and sonication. Cellular debris was removed by low speedcentrifugation. The supernatant product gave a positive result for HAVby radioimmunoassay.

B. A successful hepatitis A virus isolation was also accomplished with asecond human stool sample which was the origin of strain CR326F. Thisstool gave a positive result for hepatitis A antigen byradioimmunoassay. The stool sample was diluted and inoculated into cellculture in the same manner as described above. Immunofluorescencerevealed the first positive results at day 53 post inoculation. Thecultures were harvested at day 53 post inoculation and also gave asuccessful result for HAV antigen by radioimmunoassay.

EXAMPLE 2

A second passage of the virus from part A of Example 1 was achieved byinoculating 0.5 ml of the supernatant product from Example 1 into freshFRhK6 cell cultures that were handled comparably to those above.Fluorescence antibody examination revealed the first clear evidence ofvirus presence at day 15 post-inoculation. The cultures were harvestedat day 19 post-inoculation and the product gave a positive result forHAV by radioimmunoassay. Additional serial passage of the virus wascarried out in cell culture, i.e., passages 3, 4 and 5. By passage 5,heavy virus growth occured as early as 7-14 days and the virus harvestat day 13 gave strong positive immune adherence (IA) and radioimmuneassay (RIA) hepatitis A antigen results.

In similar manner a total of 15 serial passages of strain CR326F HAV(Ex. 1, Part B) were also accomplished. Throughout the course ofpassaging viral growth became more rapid and productive. At passage 15virus was harvested at day 8 and HAV viral antigen was detected byimmune adherence hemagglutination and radioimmunoassays.

EXAMPLE 3

Antigen, 0.05 ml, obtained as described in Example 2, was incubated withhuman convalescent hepatitis A sera, 0.02 ml of a 1:20 dilution. Themixture was incubated at 37° C. for 1 hour and then held at 4° C. for aperiod of three hours. A drop of the material was placed onto acarbon-coated, 300-mesh copper grid, and allowed to adsorb for 30seconds. The grid was then stained for 2 minutes with 2% aqueousphosphotungstic acid, pH 6.0 (adjusted with 1N KOH) and examined in aPhilips 300 electron microscope at 80 KV. After reaction with hepatitisA antibody, characteristics halos of antibody molecules were seen tosurround the numerous 27 mμ HAV particles and to bind them into animmune complex.

EXAMPLE 4

The final infected cell culture harvests obtained from Example 2,prepared under aseptic conditions, were treated with 1:4000 formalin at37° C. for 72 hours. Excess residual formalin was neutralized withsodium bisulfite. All treatments were performed under asepticconditions. The product was stored at 4° C. Subcutaneous orintramuscular injection of 4 doses of 1 ml given at 2 week intervalsinto S. mystax marmosets and guinea pigs induced circulating hepatitis Aantibody in these animals. Further, the mar mo sets were renderedresistant to challenge with virulent doses of hepatitis A virus. Thus,the ability to make an inactivated hepatitis A vaccine from HAV grown incell culture as in Examples 1 and 2 was demonstrated.

EXAMPLE 5

HAV derived as in Example 1 was serially passaged a total of 15 times inFRhK6 cell cultures at 35° C. as described in the second paragraph ofexample 2. The virus harvest from passage 15 was then successfullypropagated in fetal human diploid lung fibr oblast cell cultures (MRC5)at 35° C. A total of 8 serial in vitro passages of the virus in MRC5including 3 limit dilution passages were carried out. This yielded HAVvariant F. Variant F was found to be attenuated in virulence for bothmarmosets (S. labiatus) and chimpanzees in that intravenous inoculationof 1 ml of 1:10 dilution and 1 ml of undiluted virus, respectively, intothese animals produced serum enzyme elevations in only 1 of 6 marmosetsand none of 2 chimpanzees and only minor changes in liverhistopathology, while at the same time eliciting a hepatitis A antibodyresponse in all animals. All animals inoculated with the attenuatedvirus resist ed challenges with 1000 50% infectious doses of virulenthepatitis A virus injected intravenously. Thus the cell culture-passagedHAV variant F constitutes a live, attenuated hepatitis A vaccine.

EXAMPLE 6

Hepatitis A virus derived as described in Example 1 and the secondparagraph of Example 2 and in Example 5 was serially passed 15 times inFRhK6 cell plus 16 times in MRC5 cell cultures at 35° C. to yieldVariant F'. Variant F' was purified by 6 limit dilution passages.Variant F' was diluted 1:10 in phosphate-buffered saline (PBS) andinoculated intravenously into 6 young adult Saguinus labiatus marmosets.The animals were bled weekly for 12 weeks and again at 18 weeks. None ofthe 6 animals that received variant F' showed elevated serum isocitricdehydrogenase levels and 4 of these 6 animals developed antibody tohepatitis A. All animals that seroconverted were immune to infectionwhen challenged intravenously with virulent HAV. Two chimpanzees wereinoculated with undiluted variant F'. Neither showed evidence ofhepatitis but both developed antibody to hepatitis A. Thus, this cellculture-passaged HAV Variant F' also constitutes a live attenuatedhepatitis A vaccine.

EXAMPLE 7

A lot of live attenuated hepatitis A vaccine was made from Variant F atvirus passage level 10 in MRC5 cells. A lot of live attenuated vaccinewas made from variant F' at virus passage level 18 in MRC5 cells. Bothvaccines behaved in marmosets and chimpanzees in manner comparable tothose described in examples 5 and 6. Further, both vaccines wereclinically evaluated by parenteral administration in human volunteerswithout inducing disease. Both vaccines gave a high rate of antibodyinduction but vaccine F produced possible minor transient serum enzymeelevations in a small portion of recipients. No evidence of serum enzymeelevations was seen in recipients of vaccine F'. Thus both variants Fand F' are live attenuated hepatitis A viral vaccines for human beingsas well as for S. labiatus marmosets and chimpanzees.

What is claimed is:
 1. Propagated hepatitis A virus, not previouslypassaged in a non-human primate, propagated by cell culture of hepatitisA virus in (i) primary, continuously cultivated, or transformed cellsderived from kidney or liver tissue of human or non-human primateorigin; and/or (ii) diploid fibroblast cells derived from human ornon-human primate lung tissue; the inoculum for the cell culture beingeither a hepatitis A virus inoculum derived from a human clinicalspecimen or an inoculum from a hepatitis A virus passaged cell culturewhose initial cell culture inoculum was a hepatitis A virus inoculumderived from a human clinical specimen.
 2. Propagated hepatitis A virusaccording to claim 1 wherein the propagated virus is capable of inducinga protective immune response in humans against hepatitis A diseasewithout causing significant symptoms of hepatitis A disease.
 3. Avaccine comprising propagated hepatitis A virus according to claim
 2. 4.Propagated hepatitis A virus according to claim 2 which has beeninactivated after propagation.
 5. A vaccine comprising propagatedhepatitis A virus according to claim
 4. 6. A Propagated hepatitis Avirus, not previously passaged in a non-human primate, propagated bysequential passage comprising hepatitis A virus, in a first passagestage comprising primary, continuously cultivated, or transformed cellsderived from kidney or liver tissue of human or non-human primateorigin; and in a second passage stage comprising diploid fibroblastcells derived from human or non-human primate lung tissue; the inoculumfor which was a hepatitis A virus inoculum derived from a human clinicalspecimen or an inoculum from a hepatitis A virus passaged cell culturewhose initial cell culture inoculum was a hepatitis A virus inoculumderived from a human clinical specimen.
 7. Propagated hepatitis A virusaccording to claim 6, in which the first passage stage is in kidneycells of fetal or newborn rhesus, cynomolgus, or cercopithecus monkeyorigin.
 8. Propagated hepatitis A virus according to claim 7 in whichthe first passage stage is in FRhK6 cells.
 9. Propagated hepatitis Avirus according to claim 6 in which the second passage stage is in WI-38or MRC-5 cells.
 10. Propagated hepatitis A virus according to claim 6 inwhich the virus is capable of inducing a protective immune response inhumans against hepatitis A disease without causing significant symptomsof hepatitis A disease.
 11. A vaccine comprising propagated hepatitis Avirus according to claim
 10. 12. Propagated hepatitis A virus accordingto claim 10 which has been inactivated after propagation.
 13. A vaccinecomprising propagated hepatitis A virus according to claim
 12. 14. Acell culture comprising hepatitis A virus in (i) primary, continuouslycultivated, or transformed cells derived from kidney or liver tissue ofhuman or non-human primate origin; or in (ii) diploid fibroblast cellsderived from human or non-human primate lung tissue; the inoculum forwhich was a hepatitis A virus inoculum derived from a human clinicalspecimen without passage in a non-human primate host or an inoculum froma hepatitis A virus passaged cell culture whose initial cell cultureinoculum was a hepatitis A virus inoculum derived from a human clinicalspecimen without passage in a non-human primate host.
 15. An infectedcell culture harvest comprising hepatitis A virus which was propagatedin a cell culture of (i) primary, continuously cultivated, ortransformed cells derived from kidney or liver tissue of human ornon-human primate origin; or (ii) diploid fibroblast cells derived fromhuman or non-human primate lung tissue; using as an inoculum a hepatitisA virus inoculum derived from a human clinical specimen without passagein a non-human primate host or an inoculum from a hepatitis A viruspassaged cell culture whose initial cell culture inoculum was ahepatitis A virus inoculum derived from a human clinical specimenwithout passage in a non-human primate host.
 16. A hepatitis A vaccinecomprising attenuated, inactivated hepatitis A virus from a cell cultureof (i) primary, continuously cultivated, or transformed cells derivedfrom kidney or liver tissue of human or non-human primate origin; or(ii) diploid fibroblast cells derived from human or non-human primatelung tissue; wherein the hepatitis A virus had not been passaged througha non-human primate prior to cell culture.
 17. A process for making ahepatitis A vaccine which comprises:a) culturing hepatitis A virus,which has not been passaged through a non-human primate in (i) primary,continuously cultivated, or transformed cells derived from kidney orliver tissue of human or non-human primate origin; or in (ii) diploidfibroblast cells derived from human or non-human primate lung tissue; b)harvesting the cultured hepatitis A virus; and c) inactivating theharvested hepatitis A virus to make a vaccine.
 18. A hepatitis A vaccinecomprising hepatitis A virus passaged in a first passage stagecomprising 5 passages in cell culture of primary, continuouslycultivated, or transformed cells derived from kidney or liver tissue ofhuman or non-human primate origin; or (ii) diploid fibroblast cellsderived from human or non-human primate lung tissue; wherein thehepatitis A virus was not passaged through a non-human primate prior tocell culture.
 19. A vaccine according to claim 18 wherein the hepatitisA virus has been propagated in cell culture in a first passage stagecomprising about 5 to about 25 cell culture passages of said hepatitis Avirus from a human clinical specimen, comprising hepatitis A virus inprimary, continuously cultivated, or transformed cells derived fromkidney or liver tissue of human or non-human primate origin; followed bya second passage stage comprising at least about 5 cell culture passagesin diploid fibroblast cells derived from human or non-human primate lungtissue.
 20. A process for making an inactivated HAV vaccine whichcomprises:a) culturing hepatitis A virus, which has not been passagedthrough a non-human primate in a first passage stage comprising about 5to about 25 passages in a non-human primate kidney or liver cells and asecond passage stage comprising about 5 to about 50 passsages in humanor non-human lung fibroblast cells; b) harvesting the cultured hepatitisA virus; and c) inactivating the harvested hepatitis A virus to make avaccine.
 21. Propagated hepatitis A virus, not previously passaged in anon-human primate, propagated by cell culture passage of hepatitis Avirus in: (i) primary, continuously cultivated, or transformed cellsderived from kidney or liver of non-human primate origin; and (ii)diploid fibroblast cells derived from human lung tissue; the inoculumfor which was a hepatitis A inoculum from a human clinical specimencomprising hepatitis A virus or an inoculum from a hepatitis A viruspassaged cell culture whose initial cell culture inoculum was ahepatitis A virus inoculum derived from a human clinical specimen. 22.Propagated hepatitis A virus of claim 21 in which the propagated virusis capable of inducing a protective immune response in humans againsthepatitis A disease without causing significant symptoms of hepatitis Adisease.
 23. A vaccine comprising propagated hepatitis A virus accordingto claim
 22. 24. Propagated hepatitis A virus according to claim 22which has been inactivated after propagation.
 25. A vaccine comprisingpropagated hepatitis A virus according to claim
 24. 26. Propagatedhepatitis A virus according to claim 22 in which the first passage stageis in kidney cells of fetal or newborn rhesus, cynomologus, orcercopithecus monkey origin.
 27. Propagated hepatitis A virus accordingto claim 26 in which the first passage stage is in cells ofcercopithecus monkey origin.
 28. Propagated hepatitis A virus accordingto claim 26 in which the first passage stage is in FRhK6 cells.
 29. Thepropagated hepatitis A virus of claim 21 further comprising a secondpassage stage in diploid fibroblast cells derived from human embryoniclung tissue.
 30. The propagated hepatitis A virus according to claim 29in which the second passage stage is in WI-38 or MRC-5 cells. 31.Propagated hepatitis A virus, having been previously propagated in cellculture, said virus not having been passaged in a non-human primate,wherein said virus was propagated by cell culture in: (i) primary,continuously cultivated, or transformed cells derived from kidney orliver tissue of non-human primate origin; and (ii) diploid fibroblastcells derived from human lung tissue.
 32. Propagated hepatitis A virusaccording to claim 31 in which the propagated virus is capable ofinducing a protective immune response in humans against hepatitis Adisease without causing significant symptoms of hepatitis A disease. 33.A vaccine comprising the propagated hepatitis A virus according to claim32.
 34. Propagated hepatitis A virus according to claim 33 which hasbeen inactivated after propagation.
 35. A vaccine comprising propagatedhepatitis A virus according to claim
 34. 36. A cell culture of hepatitisA virus, wherein the hepatitis A virus had been previously propagatedin: (i) primary, continuously cultivated, or transformed cells derivedfrom kidney or liver tissue of non-human primate origin; and (ii) indiploid fibroblast cells derived from human lung tissue, wherein theinoculum for (i) was a hepatitis A virus inoculum derived from a humanclinical specimen without passage in a non-human primate host or aninoculum from a hepatitis A virus passaged cell culture whose initialcell culture inoculum was a hepatitis A virus inoculum derived from ahuman clinical specimen.
 37. Propagated virus according to claim 1wherein the human clinical specimen is a stool extract.
 38. Propagatedvirus according to claim 6 wherein the human clinical specimen is astool extract.
 39. Propagated virus according to claim 21 wherein thehuman clinical specimen is a stool extract.
 40. Propagated virusaccording to claim 31 wherein the human clinical specimen is a stoolextract.